Therapeutic Role of Tamoxifen for Triple-Negative Breast Cancer: Leveraging the Interaction Between ERβ and Mutant p53

Abstract The absence of effective therapeutic targets and aggressive nature of triple-negative breast cancer (TNBC) renders this disease subset difficult to treat. Although estrogen receptor beta (ERβ) is expressed in TNBC, studies on its functional role have yielded inconsistent results. However, recently, our preclinical studies, along with other observations, have shown the potential therapeutic utility of ERβ in the context of mutant p53 expression. The current case study examines the efficacy of the selective estrogen receptor modulator tamoxifen in p53-mutant TNBC with brain metastases. Significant increase in ERβ protein expression and anti-proliferative interaction between mutant p53 and ERβ were observed after cessation of tamoxifen therapy, with significant regression of brain metastases. This case study provides supporting evidence for the use of tamoxifen in p53-mutant, ERβ+TNBC, especially in the setting of brain metastasis.


Background
Triple-negative breast cancer (TNBC) refers to tumors that lack expression of estrogen receptor alpha (ERα), progesterone receptor (PR), and absence of HER2 amplification. Clinically, patients with TNBC have aggressive tumor biology, higher risk of recurrence, and poor prognosis. Given the absence of effective therapeutic targets, chemotherapy is the mainstay of management of TNBC but is associated with significant toxicity and low response rate, particularly in the metastatic setting.
While TNBC does not express ERα, expression of estrogen receptor beta (ERβ) in 25%-30% of TNBC offers a promising opportunity for targeted antitumorigenic therapy. 1,2 Expression of ERβ has been identified as an independent predictor of recurrence, metastasis, and mortality, and is generally regarded as a favorable clinical feature in breast cancer. ERβ1 expression, in particular, is associated with a favorable prognosis in TNBC, with reduced recurrence and improved survival outcomes. 1,3 Importantly, tamoxifen, FDA-approved medication for ERα-positive breast cancer, is also a ligand for ERβ. 4 It has been suggested that ERβ is anti-proliferative in nature, mainly based on exogenous overexpression of ERβ cDNA in cancer cell lines. [5][6][7][8][9] However, observations in other preclinical studies indicate that ERβ instead associated with proliferation in mammary epithelial cells, 10 breast cancer cells, 11 and TNBC. 2 Such inconsistent data on pro-versus anti-tumorigenic role of ERβ suggested that this protein may have bi-faceted functions. 12,13 Using si/shRNA-mediated depletion of endogenous ERβ in breast cancer cell models, we have shown that p53 status is an important determinant of the proversus anti-tumorigenic duality of ERβ in breast cancer. 14 An important characteristic of TNBC is the high prevalence (~80%) of mutations in p53. [15][16][17] Preclinical studies in breast cancer cells have shown that ERβ is capable of opposing certain pro-tumorigenic functions of mutant p53. 5,9,14,18 ERβ directly binds and antagonizes p53, 5,14,18 and importantly, wild-type versus mutant p53 status was shown to be a determinant of the pro-versus anti-tumorigenic functional duality of ERβ. 14 Our retrospective analysis of METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) data showed that combined expression of mutant p53 and high levels of ERβ in basal-like TNBC was prognostic of better breast cancerspecific patient survival. 14 Mutant p53 has been found to bind and inactivate tumor suppressor p73 when unrestricted. [19][20][21][22][23][24] ERβ was shown to bind and sequester mutant p53, resulting in the reactivation of p73. Reactivated p73 is free to promote the expression of anti-tumorigenic genes, including CDKN1A (p21) and BBC3 (PUMA), resulting in cell-cycle arrest and apoptosis. 14 Notably, we showed that tamoxifen increased ERβ-mutant p53 interaction, and p73-mutant p53 interaction was reciprocally decreased leading to the reactivation of p73 as a tumor suppressor. This finding suggested that tamoxifen's ability to inhibit the oncogenic function of mutant p53 via ERβ leading to an active p73 could be exploited for a therapeutic strategy against TNBC expressing ERβ and mutant p53. However, there has been no clinical data in support of this novel mechanism of tamoxifen function in TNBC.
Here we report an example of the efficacy of tamoxifen in TNBC. After discovery of mutant p53 variants in a metastatic nodule in October 2019, the patient's care team hypothesized that she would benefit from tamoxifen therapy based on preclinical evidence. The patient was treated with tamoxifen for approximately 2 months, after which significant antiproliferative mutant p53-ERβ interaction and a decrease in tumor burden were observed. This case contributes to the growing evidence supporting the use of tamoxifen in cases of TNBC expressing high levels of ERβ) along with mutant p53.

Patient Story
The patient is a 59-year-old G3P2 woman diagnosed with primary breast cancer and later metastasis to the brain (>10 lesions), chest wall, and neck and chest lymph nodes. Pertinent medical history includes hypertension, vitamin D deficiency, degenerative joint disease, and obesity. The patient underwent genetic testing 3 times without identification of a deleterious germline mutation in BRCA1/2, TP53, or PTEN, and had no family history of breast or ovarian malignancy.
The first malignancy was identified in July 1991 (30 years at the age of first diagnosis) when a mass was palpated in the right breast. The patient underwent a lumpectomy with axillary lymph node dissection which revealed a stage 1b, 1-cm T1bN0 grade 3, ER-(<1%)/HER2-infiltrating ductal carcinoma (IDC) with features of medullary carcinoma. She then received adjuvant cyclophosphamide, methotrexate, and fluorouracil (CMF) for 6 cycles followed by adjuvant radiation and 2 years of tamoxifen. In September 2005, a lesion in the right breast was noted on routine mammogram. Core biopsy revealed a grade 3, triple-negative (ER-/ HER2-) IDC. The patient then underwent bilateral mastectomies, during which a 0.9-cm tumor was resected from the right breast. Final pathology revealed a stage T1bNX IDC, with associated grade 3 ductal carcinoma in situ. No malignancy was identified in either the left breast, bilateral sentinel node biopsy (SNB), or the resected lymph nodes. Four cycles of adriamycin and cyclophosphamide (AC) were completed (October-December 2005), which induced menopause.
The patient's breast malignancies were stable until the fall of 2019 when she noted right submandibular swelling over a 5-to 7-week period. An MRI of the brain revealed multiple (>20) supratentorial and infratentorial brain metastases as well as intense uptake in submandibular, right hilar, subcarinal, right paratracheal, and anterior chest wall subcutaneous nodes, as outlined in Fig. 1A.

Molecular Tumor Board
A core biopsy of the right neck nodule demonstrated poorly differentiated, ER-/ PR-/ HER2 2 + (FISH 0.9) carcinoma similar to the second breast primary. Average number of HER2 copies per cell was 2.1, with HER2/cep17 ratio of 0.9. Additional specimen testing revealed GATA3+, GCDFP15-, mammaglobin-, and PDL1 + (5%) expression. PDL1 expression was evaluated via immunohistochemical staining, with determination of percent tumor area comprised of PDL1-expressing immune cells. Molecular diagnostics found mutated TP53 (i) a single nucleotide missense mutation resulting in Cys238Tyr change in the protein sequence and (ii) 18 bp/6 amino acids deletion between Pro177 and Cys182 both in the brain tumor and in the chest wall tumor tissue. Importantly, both these alterations are in the DNAbinding domain (DBD) of p53. Biopsy of the right cerebellar lesion demonstrated consistent immunohistochemistry (ER-/ PR-/ HER2 2 + (FISH 0.9) and similar genomic alterations in TP53. No copy number variants (CNVs) of known clinical significance were detected. HER2/cep17 ratio was identical to that of the neck nodule sample, with 2.0 HER2 copies per cell on average.
The patient received 2 cycles of capecitabine but had disease progression in the brain. She then received whole-brain radiotherapy. Tamoxifen was added to the radiation therapy given preclinical data demonstrating efficacy. Repeat imaging demonstrated a decrease in size and conspicuity of multiple brain lesions, as well as chest wall subcutaneous nodes (Fig. 1B). A repeat biopsy was obtained which confirmed poorly differentiated, ER-/ PR-/ HER2 negative carcinoma, TP53 mutations, as well as evidence of pharmacodynamic effect with tamoxifen.
Immunohistochemical (IHC) analysis of ERβ protein expression in the pre-tamoxifen treatment brain tumor specimen showed weak expression of the protein ( Fig. 2A) and analysis of ERβ-mutant p53 interaction in the tumor specimen by proximity ligation assay (PLA) demonstrated no interaction between the 2 proteins Fig. 2C). Contrastingly, high levels of ERβ protein expression (Fig. 1B) and increased interaction between ERβ and mutant p53 proteins (Fig. 2D) were observed in chest wall tumor tissue post-tamoxifen therapy.

Discussion
Here we report an example of efficacy of tamoxifen in metastatic mutant p53 TNBC, demonstrating both clinical activity as well as pharmacodynamic inhibition. These findings add to the evidence suggesting a role of estrogen modulator therapy in enhancing ERβ levels and the ensuing antiproliferative ERβ-mutant p53 interaction.
Tamoxifen is an anti-estrogen that selectively blocks estrogen binding to ERα through competitive inhibition. [25][26][27] Though tamoxifen is mainly used in adjuvant therapy of ERα-positive breast cancers, some clinical studies have shown  that this drug was effective in some ERα-negative tumors. 28 ERβ has been shown to be a predictive marker for favorable prognosis in response to adjuvant tamoxifen therapy of ERα-negative tumors including TNBC. 3,29-32 However, none of these studies have taken into consideration the status of p53 in the tumors. The data from the current clinical study corroborate with our preclinical studies that showed treatment of TNBC cells with tamoxifen increased interaction between ERβ and mutant p53 leading to decreased cellular proliferation. 14 A diagrammatic model depicting the mechanism underlying the effect of anti-tumor effects of Tamoxifen on TNBC is shown in Fig. 3.
In the case of brain metastases, estrogen has been shown to reduce adaptive immune response as well as phagocytic capacity of microglia via polarization to the M2 phenotype, promoting metastatic growth even in ER-negative disease. Recent mouse models have demonstrated that tamoxifen inhibits M2 polarization and brain tumor progression, suggesting tamoxifen may be an effective treatment for TNBC with brain metastases. 33 Current therapy for brain metastases in TNBC now includes monoclonal antibodies and antibody-drug conjugates such as trastuzumab-deruxtecan and Sacituzumab govitecan. 34,35 At the time this patient was undergoing treatment, these options were not standardly available. It has been hypothesized that tamoxifen in combination with monoclonal antibodies for TNBC may be more effective than either treatment in isolation, due to synergistic antiproliferative mechanisms. However, preclinical models examining tamoxifen in combination with TRA-8, a monoclonal antibody targeting death receptor 5, have demonstrated antagonistic effects. 36 Additional preclinical research on the efficacy of tamoxifen in combination with monoclonal antibodies is required before this regimen is to be considered in clinical practice. At present, we demonstrate that tamoxifen alone has significant antiproliferative effects in ERβ+, p53-mutant TNBC. Until additional research is performed, tamoxifen should be considered as a monotherapy in recurrent TNBC, particularly for patients who do not respond to antibody and/or antibody-drug therapies.
Due to the invasive nature of CNS biopsy, a chest wall sample was obtained for posttreatment evaluation of ERβ expression and interaction with mutant p53. Of note, the exact same point mutation as well as an 18 bp deletion in p53 were present both in the CNS and chest wall tumor tissues indicating that these tumors at different locations did not evolve from different clonal populations. However, because of the different cellular and tissue contexts, tumor heterogeneity between the chest wall and CNS metastases must be considered when drawing comparisons. The identification of TP53 mutation in the right cerebellar lesion prior to treatment and significant tumor regression after tamoxifen therapy allows for the conclusion of an ERβ-mutant p53-mediated antiproliferative mechanism with reasonable certainty.
While TNBC commonly recurs within 3 years of primary diagnosis, 37,38 this patient's recurrence was 14 years after the identification of the second TNBC primary in 2005. It is possible that her profound response to tamoxifen was interrelated to the indolent nature of her disease, in that the antiproliferative effects of ERβ-mutant p53 interactionare more robust in slow-growing tumors. Further research on the use of tamoxifen in more aggressive TNBC is required.
In summary, we demonstrate a potential therapeutic role of tamoxifen in a molecularly defined subset of TNBC. These novel findings require confirmation in a larger study.

Patient Update
The patient is currently doing well with no evidence of disease progression noted on last set of restaging scans (June 2022).